Amplification of nucleic acids is widely used in research, forensics, medicine and agriculture. One of the best-known amplification methods is the polymerase chain reaction (PCR), which is a target amplification method (See for example, U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159). A PCR reaction typically utilizes two oligonucleotide primers, which are hybridized to the 5′ and 3′ borders of the target sequence and a DNA polymerase which can extend the annealed primers by adding on deoxynucleoside-triphosphates (dNTPs) to generate double-stranded products. By raising and lowering the temperature of the reaction mixture, the two strands of the DNA product are separated and can serve as templates for the next round of annealing and extension, and the process is repeated.
Although PCR has been widely used by researchers, it requires thermo-cycling to separate the two DNA strands. Several isothermal target amplification methods have been developed in the past 10 years. One of them is known as Strand Displacement Amplification (SDA). SDA combines the ability of a restriction endonuclease to nick the unmodified strand of its target DNA and the action of an exonuclease-deficient DNA polymerase to extend the 3′ end at the nick and displace the downstream DNA strand. The displaced strand serves as a template for an antisense reaction and vice versa, resulting in exponential amplification of the target DNA (See, for example, U.S. Pat. Nos. 5,455,166 and 5,470,723). In the originally-designed SDA, the DNA was first cleaved by a restriction enzyme in order to generate an amplifiable target fragment with defined 5′ and 3′-ends but the requirement of a restriction enzyme cleavage site limited the choice of target DNA sequences (See for example, Walker et. al., Proc. Natl. Acad. Sci. USA 89:392-396 (1992)). This inconvenience has been circumvented by the utilization of bumper primers which flank the region to be amplified (Walker et al. supra (1992)). SDA technology has been used mainly for clinical diagnosis of infectious diseases such as chlamydia and gonorrhea. One of the most attractive feature of SDA is its operation at a single temperature which circumvents the need for expensive instrumented thermal cycling. However, SDA is inefficient at amplifying long target sequences.
A second isothermal amplification system, Transcription-Mediated Amplification (TMA), utilizes the function of an RNA polymerase to make RNA from a promoter engineered in the primer region, and a reverse transcriptase, to produce DNA from the RNA templates. This RNA amplification technology has been further improved by introducing a third enzymatic activity, RNase H, to remove the RNA from cDNA without the heat-denaturing step. Thus the thermo-cycling step has been eliminated, generating an isothermal amplification method named Self-Sustained Sequence Replication (3SR) (See, for example, Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878 (1990)). However, the starting material for TMA and 3SR is limited to RNA molecules.
A third isothermal target amplification method, Rolling Circle Amplification (RCA), generates multiple copies of a sequence for the use in in vitro DNA amplification adapted from in vivo rolling circle DNA replication (See, for example, Fire and Xu, Proc. Natl. Acad. Sci. USA 92:4641-4645 (1995); Lui, et al., J. Am. Chem. Soc. 118:1587-1594 (1996); Lizardi, et al., Nature Genetics 19:225-232 (1998), U.S. Pat. Nos. 5,714,320 and 6,235,502). In this reaction, a DNA polymerase extends a primer on a circular template generating tandemly linked copies of the complementary sequence of the template (See, for example, Kornberg and Baker, DNA Replication, W.H. Freeman and Company, New York (2nd ed. (1992)). Recently, RCA has been further developed in a technique, named Multiple Displacement Amplification (MDA), which generates a highly uniform representation in whole genome amplification (See, for example, Dean et. al., Proc. Natl. Acad. Sci. USA 99:5261-5266 (2002)).
Additional nucleic acid amplification methods include Ligase Chain Reaction (LCR), which is a probe amplification technology (See, for example, Barany, Proc. Natl. Acad. Sci. USA 88:189-193 (1991)); and U.S. Pat. No. 5,494,810), and branched DNA (bDNA) technology (Horn et al., Nucleic Acids Res. 25:4842-4849 (1997)), which is a signal amplification technology.
The amplification methods mentioned above all have their limitations. For example, PCR and LCR require a thermocycler with associated instrumentation. Except for PCR, none of the other target amplification methods are capable of amplifying DNA targets having sufficient length to be useful for cloning genes and analysis of virulence factors and antibiotic resistant genes. Although PCR is able to amplify a target up to 10-20 kb, high mutation rates may limit the use of PCR-amplified products (Cline et al., Nucleic Acids Res. 24, 3546-3551 (1996)). Thus, to minimize the problem, a high-fidelity amplification method for long targets is needed. In addition, all present amplification methods require prior heat denaturation and annealing steps to produce primed templates for DNA polymerases. This adds extra time to the amplification process.
The potential uses for nucleic acid amplification techniques continues to grow. For example, nucleic acid arrays frequently utilize large numbers of amplification reactions. Detection of environmental contamination places demands on sensitivity and analytic power of diagnostic tests that include nucleic acid amplification procedures. Consequently, improvements in amplification methodology are desirable.